Detector and method for autoradiography

ABSTRACT

There is disclosed an autoradiography system which includes an autoradiography sample, a substantially radioisotope free microchannel plate (MCP), and an MCP signal collection means, wherein the MCP directly measures beta particles from radioisotopes within the sample.

This invention relates to the field of autoradiography.

Film autoradiography, wherein radiological grade X-ray film is used torecord two dimensional spatial beta-radioactivity patterns, is astandard procedure in bio-medical research. The sample may be present asa thin tissue section or dried electrophoresis gel, such as is used inDNA ‘fingerprinting’ techniques. Film autoradiography offersconsiderable advantages, such as ready availability, low capital cost,very high intrinsic spatial resolution, but balanced against theseadvantages are some serious disadvantages, principally the very longexposure times—days, weeks or even months—required to produce adevelopable image. The long exposure times are a direct result of thevery low activity typically present per image feature (an image featurebeing, for example, a discrete band in a DNA sequence). Furtherdisadvantages associated with the use of film are the non-linearresponse and limited dynamic range associated therewith, and also thefact that post-processing (e.g. microdensitometry) is required in orderto produce quantitative data on radioisotope uptake.

In order to alleviate the aforementioned disadvantages, a number ofelectronic position sensitive detectors have been employed in place ofX-ray film. These techniques are:

i) Multiwire and other forms of gas proportional counters;

ii) Photoelectric image intensifiers, for instance comprising ascintillator layer, fibre optic faceplate, S20 optical photocathode andmicrochannel plate gain stage;

iii) Photostimulable phosphors or “Image Plates”;

iv) Fibre-optic coupled cooled CCDs.

However, each of these techniques suffers from significant drawbacks.Techniques i)-iii) have large associated capital costs. Gas countersoffer spatial resolutions of the order of 1 mm and are virtually unableto detect tritium (³H) since the low energy ³H betas (endpoint energy18.6 KeV) cannot penetrate the counter window. Tritium detection byimage intensifier, image plates and CCD arrangements is generallyinefficient because of the small optical signal developed in the inputlayer. Often a fibre-optic demagnifying taper or lens is required toreduce the desired working field approximately (100 cm² area) to thevery much smaller active area of the output sensor resulting in furtherloss of signal. The optical transmission of a 5:1 taper is at most a fewpercent.

The present invention overcomes the abovementioned problems by employinga low noise microchannel plate detector to perform autoradiography.

According to a first aspect of the invention there is provided adetector for autoradiography, the detector comprising a substantiallyradioisotope free microchannel plate (MCP) detector, the dimensions ofthe active area of said detector being substantially comparable with, orgreater than, the working field of the autoradiography sample, and MCPsignal collection means.

The dark count of the MCP detector may be less than 0.15counts.cm⁻².s⁻¹.

The MCP detector may comprise potassium and rubidium free glass.

The MCP detector body parts may comprise PCTFE.

The MCP detector may be shielded against background gamma rays.

The detector may further comprise a resistive anode readout element.

The front plate of the MCP detector may be held at, or substantially at,ground potential.

The detector may measure beta particles emitted by tritium.

According to a second aspect of the invention there is provided a methodfor performing autoradiography comprising the steps of:

providing an autoradiography sample; and

measuring beta particles emitted by radioisotopes within said samplewith a substantially radioisotope free MCP detector, the dimensions ofthe active area of said detector being substantially comparable with, orgreater than, the working field of the autoradiography sample, and MCPsignal collection means.

Methods and apparatus for performing autoradiography will now describedwith reference to the accompanying drawings, in which:

FIG. 1 shows a ³H autoradiogram of a tritium standard;

FIG. 2 shows a graph of detection response against tritium cellactivity;

FIG. 3 shows a ³H autoradiogram of rat lung tissue;

FIG. 4 is an optical light photograph of rat lung tissue;

FIGS. 5(a)-5(d) shows ³⁵S autoradiograms at a variety of exposure times;

FIG. 6 shows the ³⁵S autoradiogram after an exposure time of 8000 S;

FIG. 7 shows the ³⁵S autoradiogram after an exposure of approximately 16hours; and

FIG. 8 shows a ¹⁴C autoradiogram of a rat whole-body tissue slice.

FIG. 9 shows a typical mounting arrangement for mounting themicrochannel plate used in the system of the invention; and is also asimplified view of the radionuclide detection which occurs in the systemof the invention.

The invention comprises a detector for autoradiography, the detectorcomprising a substantially radioisotope free MCP detector, thedimensions of the active area of said detector being substantiallycomparable with, or greater than, the working field of theautoradiography sample, and MCP signal collection means. In the exampledescribed below two pairs of chevron MCP detectors were employed as MCPdetectors. One pair of MCPs had a large active area of 93 mm×93 mm,whilst the other pair had a smaller active area (30 mm diameter). MCPcharacteristics are detailed in Table 1.

The detectors employ two low noise glass MCPs (Philips Photonics, Brivela Gaillarde Cedex, France) producing a dark count of less than 0.15counts cm⁻²s⁻¹. The glass is potassium and rubidium free: in fact, themain source of detector background signal in “standard” lead silicateglass MCPs is due to the ⁴⁰K β emission of constituent potassium. Alldetector body parts are manufactured from PCTFE (Poly-chloro tetrafluoro ethylene, Fluorocarbon Company Ltd, Hertford, United Kingdom),since conventional materials, such as Macor, contain potassium. Darknoise rates for both of the detectors employed were 0.12 counts.cm⁻² s⁻¹above a discriminator level of 0.05× peak detector gain. Such low darkcount rates are important given the inherently low signal levelsassociated with autoradiography samples. The dark count level may bereduced still further by passive lead shielding of the detector againstthe 1.5 MeV ⁴⁰K gamma radiation which constitutes the bulk of theresidual background. This radiation emanates from the concrete of thelaboratory walls and floors.

A resistive anode readout element was employed at the chevron output asMCP signal collection means (see, for example, G W Fraser, M A Barstowand J F Pearson, Nucl. Instr. Meth., A273 (1988) 667).

The use of low noise MCP detectors whose active areas are comparable tothe working field of the sample permits the detector to be placed veryclose to, or actually directly onto, the sample. This provides interalia a full image, good sensitivity and high spatial resolution.

The mounting of the sample with respect to the microchannel plate can beby simple proximity and can be explained with reference to theaccompanying drawing, FIG. 9.

EXAMPLE 1 TRITIUM STANDARD

A tritium standard source (American Radiolabeled Chemicals Inc., StLouis, USA) was mounted 0.5 mm from the 93 mm×93 mm active areadetector. The detector and the standard were then placed under vacuum(<10⁻⁶ mbar operating pressure). FIG. 1 shows the resulting ³Hautoradiogram, accumulated over 20 hours. The standard has fourteen 7×5mm² tritium loaded wax “cells” of logarithmically decreasing activities(quoted as 466.5-0.0 μCi g⁻¹) present in “two lanes” of seven cells.Eleven cells are visible above the background in FIG. 1. Two points tobe noted are that the cell activities displayed in FIG. 1 have beencorrected for the age of the standard, and that image intensities aredisplayed on a log₁₀ scale. The corrected activity of the least intensecell to be detected is 0.39 μCi g⁻¹, and therefore the sensitivity ofthe large area MCP detector to ³H is at least 0.39 μCi g⁻¹ in 20 hours.

In this example—and indeed in the other examples as described herein—anegative potential of approximately 4.5 kV is applied to the front plate(with the readout element at ground). Such a large negative potentialwill repel a large fraction of the low energy ³H betas. Calculationssuggest that a factor of two increase in beta count rate would result ifthe detector were operated with the front plate at zero potential andthe readout element at high positive potential.

The spatial resolution was estimated, from the edge response function ofthe most intense cell, to be 400 μm FWHM at the 0.5 mm standard-MCPseparation. However, the intrinsic resolution of the large areadetector, previously determined from X-ray measurements, isapproximately 80 μm. The spatial resolution of detectors which employresistive anode readout scales inversely with resistive anode sidelength. Therefore, it is possible to improve spatial resolution for ³Hbeta detection by

(a) reducing the size of the detector and hence the size of theresistive anode required, and

(b) operating at reduced sample-detector separations.

The size of the detector required for a given application is, of course,dependent on the size of the sample. This is because it is necessarythat, if a full sample image is to be obtained, the dimensions of theactive area of the detector are at least substantially comparable withthe working field of the sample. Therefore,. smaller samples permit theuse of smaller detectors (see Example 2). Operating at reducedsample-detector separations will also have the effect of increasingsignal levels. In fact, elimination of the (beta velocity retarding)negative input potential would also serve to improve spatial resolution.

FIG. 2 shows the linearity of the detector response derived from theintensities of the images of the tritium standard cells. The maximumcount rate of the brightest cell corresponds to a count rate per channelof just 7.4×10⁻⁵.s⁻¹. The clear implication is that the responselinearity demonstrated in FIG. 2 to hold over three orders of magnitudein fact extends to six orders of dynamic range.

EXAMPLE 2 ³H LABELLED BIOLOGICAL SAMPLES

³H labelled biological samples were imaged. The samples were rat lungslices which had been labelled with tritiated Putrescine(1,4-diaminobutane). Semi thin tissue sections (1 μm thick) wereembedded, after preprocessing, in Araldite (RTM) as per preparation forconventional contact emulsion autoradiography. The smaller MCP detector(active area 30 mm diameter) was employed in order to substantiallymatch with the rat time sample size whilst improving spatial resolution.The sample was mounted onto a glass cover slip and then placed in directcontact with the MCP input surface.

FIG. 3 shows the resulting image from the lung tissue slice, after anaccumulation period of 76 hours. For comparison FIG. 4 shows an opticalphotograph of the sample, obtained using a binocular microscope. In bothFIGS. 3 and 4 air passages 20,22, both perpendicular 20 and in the plane22 of the sample, are clearly visible. There is no tritium uptake inthese regions.

An upper limit to the image resolution is estimated as 100 μm—a moreaccurate measurement was not possible since the edge of the sample, fromoptical microscope inspection, is only very approximately straight.However, the improvement over the spatial resolution of the image ofExample 1 confirms the expectation that reduced detector dimensions andreduced sample-detector separations would lead to an enhancement inimage resolution.

The activity of the tissue sample was unknown, but from the known MCPbeta detection efficiency (50%) the measured noise subtracted MCP countrate (0.059 counts s⁻¹), the sample thickness (1 μm) and an assumedsample density of 1 g cm³¹ ³ an estimated activity of approximately 0.52μCi g⁻¹ is obtained.

EXAMPLE 3 ³⁵S DETECTION

³⁵S beta autoradiograms were recorded with the large active area MCPdetector. The autoradiograms are of a sample produced by gelelectrophoresis of slime mould DNA. The gel was dried and, forcomparison purposes, was first placed in contact with conventional X-rayfilm in a 36 hour exposure. The gel was then mounted in vacuum inintimate contact with the front face of the input channel plate of thechevron pair and the ³⁵S beta image recorded, over a total period of 16hours

In FIGS. 5 to 7 are presented the 35S beta autoradiograms, recorded at avariety of exposure times with the MCP detector. In all cases the imagesize is 93×93 mm². It should be noted that: i) FIGS. 5 to 7 are in goodagreement with the results of the X-ray film experiment; ii) the MCPimages are in a digital form which has been image processed—in fact, theraw 2048×2048 file had to be binned to 512×512 for display of the fullfield, with a consequent loss of resolution; iii) the faint diagonalline on all of FIGS. 5 to 7 is an ADC artefact; and iv) the ‘pincushion’ distortion apparent at two of the sides of the images caneasily be removed by further software processing. FIGS. 5a, 5 b, 5 c and5 d show the MCP image after exposure times of 1000, 2000, 4000 and 8000seconds, respectively. The 8000s image is also shown, on a larger scale,in FIG. 6, whilst FIG. 7 shows the final image after an exposure time ofapproximately 57600 s (16 hours). The beta pattern 50 corresponding tothe most active of the four gel “lanes” is apparent after a mere 1000 s,and clearly readable after only 8000 s (2.2 hours). The full image ofFIG. 7 displays a very large dynamic range between pattern 50 and theother patterns 60, 70, 80. The undulation of the patterns 50-80 is real,and is confirmed by the film contact print.

EXAMPLE 4 ¹⁴C LABELLED BIOLOGICAL SAMPLES

A further demonstration is provided by FIG. 8, which shows a ¹⁴Cautoradiogram (end-point energy 156 KeV) generated by MCP detection of asample of ¹⁴C labelled rat tissue after an exposure time of 105 minutes.

MCP detection offers high spatial resolution—certainly compared to gascounter systems. For example, the spatial resolution obtained with thepresent invention, 100 μm or less, compares to a “best” spatialresolution of approximately 500 μm obtained with the optical avalanchechamber type gas proportional counter. Image plates can be used forimaging tritium, but require special plates. A typical quoted minimumdetectable activity for ³H is 1.67 Bq mm⁻² hr⁻¹, which is a factor of 80poorer than the present invention. The large dimensions of the MCPdetector (in relation to the size of the sample) result in a field ofview greater than that of current fibre-optic coupled CCD systems,whilst the overall detection efficiency of the (single stage) MCPdetection process is certainly much higher. If the activity of a singleimage element is R, then the MCP signal count rate from that elementwill be approximately R/4 (Babenkov et al, Nucl. Instr. Meth., A252(1986) 83) within a factor of approximately 2 of the ideal. Furthermorethe nature of the MCP output is suited for digitisation andstorage/processing on computer. The electron detection efficiency of thewindowless 12.5 μm pore plates, as measured by Babenkov et al, is likelyto be in excess of 50% not only for ³H beta radiation (end-point energy18.6 KeV) but also for ³²p (end-point energy 1.7077 MeV) and ³⁵S(end-point energy 168 KeV, average energy 48.8 KeV) beta radiation.Detection of ³⁵S and ³²P betas is important for electrophoresis gelapplications, whilst the detection of tritium betas is of particularimportance since it permits autoradiography of tissue slices.

TABLE 1 MCP parameters Parameter Value Thickness (L) 1.5 mm Channeldiameter (D) 12.5 μm Channel pitch 15.0 μm L:D 120:1 Channel bias angle6°

What is claimed is:
 1. An autoradiography system, comprising: anautoradiography sample; a substantially radioisotope free microchannelplate (MCP) detector having an active area, the dimensions of the activearea of said detector being substantially comparable with, or greaterthan, the working field of the autoradiography sample; MCP signalcollection means; and wherein the MCP detector directly measures betaparticles emitted by radioisotopes within the autoradiography sample. 2.An autoradiography system according to claim 1, in which the MCPdetector has a characteristic dark count, the dark count of the MCPdetector being less than 0.15 counts.cm⁻².s⁻¹.
 3. An autoradiographysystem according to claim 1, in which the MCP detector comprisespotassium and rubidium free glass.
 4. An autoradiography systemaccording to claim 1, in which the MCP detector is comprised of bodyparts, the body parts being formed of poly-chloro tetra fluoroethylene.5. An autoradiography system according to claim 1, in which the MCPdetector is shielded against background gamma rays.
 6. Anautoradiography system according to claim 1, in which the MCP signalcollection means comprises a resistive anode readout element.
 7. Anautoradiography system according to claim 1, in which the MCP detectorhas a front plate, the front plate being held at, or substantially at,ground potential.
 8. An autoradiography system according to claim 1, inwhich beta particles emitted by tritium are detected.
 9. Theautoradiography system of claim 1, wherein the MCP detector is placedvery close to, or in actual contact with, the autoradiography sample.10. A method for performing autoradiography comprising the steps of:providing an autoradiography sample; directly measuring beta particlesemitted by radioisotopes within said sample with a substantiallyradioisotope free MCP detector having an active area, the dimensions ofthe active area of said detector being substantially comparable with, orgreater than, the working field of the sample, and MCP signal collectionmeans.
 11. The method of claim 10, in which the MCP detector has acharacteristic dark count, the dark count of the MCP detector being lessthan 0.15 counts.cm⁻².s⁻¹.
 12. The method of claim 10, in which the MCPdetector comprises potassium and rubidium free glass.
 13. The method ofclaim 10, in which the MCP detector is comprised of body parts, the bodyparts being formed of poly-chloro tetra fluoroethylene.
 14. The methodof claim 10, in which the MCP detector is shielded against backgroundgamma rays.
 15. The method of claim 10, in which the MCP signalcollection means comprises a resistive anode readout element.
 16. Themethod of claim 10, in which the MCP detector has a front plate, thefront plate being held at, or substantially at, ground potential. 17.The method of claim 10, in which beta particles emitted by tritium aredetected.
 18. The method system of claims 10, wherein the MCP detectoris placed very close to, or in actual contact with, the autoradiographysample.